β-Carotene interference with UVA-induced gene expression by multiple pathways

UVA exposure causes skin photoaging by singlet oxygen (1O2)-mediated induction of matrix metalloproteases (MMPs). We assessed whether β-carotene, a carotenoid known as 1O2 quencher and retinoic acid (RA) precursor, interferes with UVA-induced gene regulation and prevents UVA-induced gene regulation in HaCaT human keratinocytes. HaCaT cells accumulated β-carotene in a time- and dose-dependent manner. UVA irradiation massively reduced the cellular β-carotene contents. β-Carotene suppressed UVA induction of MMP-1, MMP-3, and MMP-10 - three major MMPs involved in photoaging. HaCaT cells produced weak retinoid activity from β-carotene, as demonstrated by mild up-regulation of retinoid receptor RARβ and activation of an RARE-dependent reporter gene. Of the 568 UVA-regulated genes, β-carotene reduced the UVA effect for 143, enhanced it for 180, and did not interact with UVA for 245 genes. The pathways regulated β-carotene in interaction with UVA were characterized by genes involved in growth factor signaling, stress response, apoptosis, cell cycle, extracellular matrix (ECM) degradation, tanning, and inflammation. In conclusion, β-carotene at physiological concentrations interacted with UVA effects by multiple mechanisms that included, but were not restricted to, 1O2 quenching. With our results, we provide a mechanistic basis for the long-known and clinically established photoprotective effects of β-carotene in human skin.

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Abstract 3515: VEGF And Peroxynitrite Interplay Regulates Cerebral Angiogenic Mediators In Diabetes

Stroke ◽

10.1161/str.43.suppl_1.a3515 ◽

2012 ◽

Vol 43 (suppl_1) ◽

Author(s):

Roshini Prakash ◽

Azza B El-Remessy ◽

Somanath P Shenoy ◽

Susan C Fagan ◽

Adviye Ergul

Keyword(s):

Kinase Inhibitor ◽

Vascular Complications ◽

Neutralizing Antibody ◽

Therapeutic Angiogenesis ◽

Tube Formation ◽

Matrix Metalloproteases ◽

Diabetic Group ◽

Growth Factor Signaling ◽

Dependent Manner ◽

Angiogenic Potential

Background: Diabetes targets micro and macrovascular endothelium differently leading to vascular complications. Although there are extensive studies on retinal and coronary neovascularization, little is known about cerebral neovascularization. Growing evidence suggests that reactive oxygen and nitrogen species such as peroxynitrite can interact in growth factor signaling and mediate VEGF’s angiogenic properties. This study tests the hypothesis that diabetes stimulates cerebral angiogenesis via activation of VEGF and matrix metalloproteases (MMP) in a peroxynitrite-dependent manner. Methods: Cerebral microvascular endothelial cells (CMEC) were isolated using immunomagnetic beads from control and diabetic Goto-Kakizaki (GK) rats. Cell proliferation, migration and tube formation assays were used as the indices of angiogenic potential. Protein expression and activity were determined using immunoblotting, slot blots and zymography techniques. Results: Angiogenic potential was pronounced in the diabetic group and significantly inhibited by peroxynitrite decomposition catalyst FeTPPS (2.5µM), MMP inhibitor minocycline (50µg/ml) and src kinase inhibitor PP2(1µM) compared to control. Diabetic endothelial conditioned media pronounced angiogenic potential in control CMEC while VEGF neutralizing antibody abrogated this response. Diabetes increased the expression of soluble VEGF A isoforms and activity of cognate receptors. Basal levels of phospho-c-src and peroxynitrite were also increased in the diabetic group. Secreted active MMP-2 and pro-MMP-2 was lower in diabetes while the cellular active MMP-2 and MT1-MMP was higher compared to control. VEGF (30ng/ML) stimulation activated its cognate receptor biophysically, dysregulated c-src activity and increased tyrosine nitration in diabetes. Peroxynitrite inhibition reduced VEGFR2, phosphor-c-src and MT1-MMP even in the presence of VEGF stimulation. Conclusion: Peroxynitrite interacts with VEGF signaling in diabetes and dysregulates c-src and MMPs. Understanding cerebral neovascularization mechanisms will not only open new paths towards developing therapeutic angiogenesis but also prevention of hemorrhagic complications of stroke. N=3-8 * p≤ 0.05, ** p≤ 0.005, *** p≤ 0.001.

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Anti-Photoaging and Anti-Melanogenesis Effects of Fucoidan Isolated from Hizikia fusiforme and Its Underlying Mechanisms

Marine Drugs ◽

10.3390/md18080427 ◽

2020 ◽

Vol 18 (8) ◽

pp. 427 ◽

Cited By ~ 1

Author(s):

Lei Wang ◽

Jae-Young Oh ◽

Young-Sang Kim ◽

Hyo-Geun Lee ◽

Jung-Suck Lee ◽

...

Keyword(s):

Human Keratinocytes ◽

Mitogen Activated Protein Kinase ◽

Dependent Manner ◽

Hacat Cells ◽

Concentration Dependent Manner ◽

B16f10 Cells ◽

Hizikia Fusiforme ◽

Mitogen Activated Protein ◽

Underlying Mechanisms ◽

Induced Apoptosis

Previous studies suggested that fucoidan with a molecular weight of 102.67 kDa, isolated from Hizikia fusiforme, possesses strong antioxidant activity. To explore the cosmeceutical potential of fucoidan, its anti-photoaging and anti-melanogenesis effects were evaluated in the present study. The anti-photoaging effect was investigated in ultraviolet (UV) B-irradiated human keratinocytes (HaCaT cells), where fucoidan effectively reduced the intracellular reactive oxygen species level and improved the viability of the UVB-irradiated cells without any cytotoxic effects. Moreover, fucoidan significantly decreased UVB-induced apoptosis in HaCaT cells by regulating the protein expression of Bax, Bcl-xL, PARP, and Caspase-3 in HaCaT cells in a concentration-dependent manner. The anti-melanogenesis effect of fucoidan was evaluated in B16F10 melanoma cells that had been stimulated with alpha-melanocyte-stimulating hormone (α-MSH), and fucoidan treatment remarkably inhibited melanin synthesis in α-MSH-stimulated B16F10 cells. Further studies indicated that fucoidan significantly suppressed the expression of tyrosinase and tyrosinase-related protein-1 and -2 (TRP-1 and-2) in B16F10 cells by down-regulating microphthalmia-associated transcription factor (MITF) through regulation of the ERK–MAPK (extracellular signal regulated kinase-mitogen activated protein kinase) pathway. Taken together, these results suggest that fucoidan isolated from H. fusiforme possesses strong anti-photoaging and anti-melanogenesis activities and can be used as an ingredient in the pharmaceutical and cosmeceutical industries.

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TgROP18 Targets IL20RB for Host Defence Related-STAT3 Activation During T. gondii Infection

10.21203/rs.3.rs-16783/v1 ◽

2020 ◽

Author(s):

Ling Kong ◽

Dan Jiang ◽

Cheng He ◽

Jing Xia ◽

Haixia We ◽

...

Keyword(s):

Host Cell ◽

Human Keratinocytes ◽

Host Cells ◽

Receptor Protein ◽

Dependent Manner ◽

Hacat Cells ◽

Double Knockout ◽

Protein Protein Interaction ◽

Stat3 Phosphorylation ◽

Sds Page

Abstract Background: Toxoplasma gondii is an opportunistic protozoan infecting almost one third of the world’s population. T. gondii rhoptry protein 18 (TgROP18) is a key virulence factor determining parasite’s acute virulence and secreted into host cells during infection. We previously identified the interaction of TgROP18 and host cell immune-related receptor protein IL20RB, and observed the activation of STAT3 in human keratinocytes (HaCaT) cells infected by the rop16 knockout RH strain, though TgROP16 is regarded responsible for host STAT3 activation during T. gondii invasion. Therefore, we hypothesize TgROP18 can activate host STAT3 through binding to IL20RB.Methods: CRISPR-CAS9 technology was used to generate the ROP16 and ROP8 double knockout RH strain, RH-∆rop16∆rop18. SDS-PAGE and western blot was used to detect STAT3 activation in different T. gondii tachyzoites infected, recombinant ROP18 or IL-20 treated HaCaT cells with high endogenous IL20RB expression. FRET and Co-immunoprecipitation (Co-IP) was used to detect the protein-protein interaction. Results: We observed that TgROP18 was involved in a synergic activation of host JAK/STAT3 pathway together with TgROP16 in human HaCaT cells infected with T. gondii or treated with recombinant TgROP18 protein, stimulating host proinflammatory immune responses such as expression of TNF-a. The effect of recombinant ROP18 on STAT3 phosphorylation was presented in a dose-dependent manner. Additionally, TgROP18 was identified to target IL20RB on its extracellular domain. When we treated different cell lines with the recombinant ROP18, STAT3 phosphorylation could only be observed in the cells with endogenous IL20RB expression, such as HaCaT cells. Conclusions: These findings indicated that TgROP18-IL20RB interaction was involved in STAT3 activation upon T. gondii invasion, which is associated with host cell defence.

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Effects of Wannachawee Recipe with Antipsoriatic Activity on Suppressing Inflammatory Cytokine Production in HaCaT Human Keratinocytes

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2017/5906539 ◽

2017 ◽

Vol 2017 ◽

pp. 1-12 ◽

Cited By ~ 5

Author(s):

Mingkwan Na Takuathung ◽

Ariyaphong Wongnoppavich ◽

Pornsiri Pitchakarn ◽

Ampai Panthong ◽

Parirat Khonsung ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Human Keratinocytes ◽

Convincing Evidence ◽

Potential Candidate ◽

Dependent Manner ◽

Hacat Cells ◽

Concentration Dependent Manner ◽

Folk Remedy ◽

Anti Inflammatory

Psoriasis is a chronic inflammatory and immune-mediated skin disease. The pathogenesis involves T cells activation via the IL-23/Th17 axis. Conventional treatments of psoriasis have adverse events influencing patients’ adherence. Wannachawee Recipe (WCR) has been effectively used as Thai folk remedy for psoriasis patients; however, preclinical evidence defining how WCR works is still lacking. This study defined mechanisms for its antiproliferation and anti-inflammatory effects in HaCaT cells. The cytotoxicity and antiproliferation results from SRB and CCK-8 assays showed that WCR inhibited the growth and viability of HaCaT cells in a concentration-dependent manner. The distribution of cell cycle phases determined by flow cytometry showed that WCR did not interrupt cell cycle progression. Interestingly, RT-qPCR revealed that WCR significantly decreased the mRNA expression of IL-1β, IL-6, IL-8, IL-17A, IL-22, IL-23, and TNF-α but induced IL-10 expression in TNF-α- and IFN-γ-induced HaCaT cells. At the protein level determined by ELISA, WCR significantly reduced the secretion of IL-17A, IL-22, and IL-23. The WCR at low concentrations was proved to possess anti-inflammatory effect without cytotoxicity and it did not interfere with cell cycle of keratinocytes. This is the first study to provide convincing evidence that WCR is a potential candidate for development of effective psoriasis therapies.

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TgROP18 Targets IL20RB for Host-Defense-Related-STAT3 Activation During T. gondii Infection

10.21203/rs.3.rs-16783/v2 ◽

2020 ◽

Author(s):

Ling Kong ◽

Dan Jiang ◽

Cheng He ◽

Jing Xia ◽

Haixia We ◽

...

Keyword(s):

Host Cell ◽

Human Keratinocytes ◽

Host Cells ◽

Receptor Protein ◽

Dependent Manner ◽

Hacat Cells ◽

Double Knockout ◽

Protein Protein Interaction ◽

Cell Defense ◽

Stat3 Phosphorylation

Abstract Background: Toxoplasma gondii is an opportunistic protozoan infecting almost one -third of the world’s population. T. gondii rhoptry protein 18 ( Tg ROP18) is a key virulence factor determining the parasite’s acute virulence and is secreted into host cells during infection. We previously identified the interaction of Tg ROP18 and host cell immune-related receptor protein IL20RB, and observed the activation of STAT3 in human keratinocytes (HaCaT) cells infected by the rop16 knockout RH strain, though Tg ROP16 is regarded as being responsible for host STAT3 activation during T. gondii invasion. Therefore, we hypothesize Tg ROP18 can activate host STAT3 through binding to IL20RB. Methods: CRISPR-CAS9 technology was used to generate the ROP16 and ROP8 double knockout RH strain, RH-∆rop16∆rop18. SDS-PAGE and Western blot were used to detect STAT3 activation in different HaCaT cells with high endogenous IL20RB expression treated with T. gondii tachyzoites infection, recombinant ROP18, or IL-20 . FRET and co-immunoprecipitation (Co-IP) was used to detect the protein-protein interaction. Results: We observed that TgROP18 was involved in a synergic activation of the host JAK/STAT3 pathway together with TgROP16 in human HaCaT cells infected with T. gondii or treated with recombinant TgROP18 protein, stimulating host proinflammatory immune responses such as expression of TNF-a. The effect of recombinant ROP18 on STAT3 phosphorylation was presented in a dose-dependent manner. Additionally, TgROP18 was identified to target IL20RB on its extracellular domain. When we treated different cell lines with the recombinant ROP18, STAT3 phosphorylation could only be observed in the cells with endogenous IL20RB expression, such as HaCaT cells. Conclusions: These findings indicate that TgROP18-IL20RB interaction upon T. gondii invasion was involved in STAT3 activation, which is associated with host cell defense.

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TgROP18 Targets IL20RB for Host-Defense-Related-STAT3 Activation During T. gondii Infection

10.21203/rs.3.rs-16783/v3 ◽

2020 ◽

Author(s):

Ling Kong ◽

Dan Jiang ◽

Cheng He ◽

Jing Xia ◽

Haixia We ◽

...

Keyword(s):

Host Cell ◽

Human Keratinocytes ◽

Host Cells ◽

Receptor Protein ◽

Dependent Manner ◽

Hacat Cells ◽

Double Knockout ◽

Protein Protein Interaction ◽

Cell Defense ◽

Stat3 Phosphorylation

Abstract Background: Toxoplasma gondii is an opportunistic protozoan infecting almost one-third of the world’s population. T. gondii rhoptry protein 18 (TgROP18) is a key virulence factor determining the parasite’s acute virulence and is secreted into host cells during infection. We previously identified the interaction of TgROP18 and host cell immune-related receptor protein IL20RB, and observed the activation of STAT3 in human keratinocytes (HaCaT) cells infected by the rop16 knockout RH strain, though TgROP16 is regarded as being responsible for host STAT3 activation during T. gondii invasion. Therefore, we hypothesize TgROP18 can activate host STAT3 through binding to IL20RB.Methods: CRISPR-CAS9 technology was used to generate the ROP16 and ROP18 double knockout RH strain, RH-∆rop16∆rop18. SDS-PAGE and Western blot were used to detect STAT3 activation in different HaCaT cells with high endogenous IL20RB expression treated with T. gondii tachyzoites infection, recombinant ROP18, or IL-20. FRET and co-immunoprecipitation (Co-IP) was used to detect the protein-protein interaction. Results: We observed that TgROP18 was involved in a synergic activation of the host JAK/STAT3 pathway together with TgROP16 in human HaCaT cells infected with T. gondii or treated with recombinant TgROP18 protein, stimulating host proinflammatory immune responses such as expression of TNF-a. The effect of recombinant ROP18 on STAT3 phosphorylation was presented in a dose-dependent manner. Additionally, TgROP18 was identified to target IL20RB on its extracellular domain. When we treated different cell lines with the recombinant ROP18, STAT3 phosphorylation could only be observed in the cells with endogenous IL20RB expression, such as HaCaT cells. Conclusions: These findings indicate that TgROP18-IL20RB interaction upon T. gondii invasion was involved in STAT3 activation, which is associated with host cell defense.

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Microgravity Induces Transient EMT in Human Keratinocytes by Early Down-Regulation of E-Cadherin and Cell-Adhesion Remodeling

Applied Sciences ◽

10.3390/app11010110 ◽

2020 ◽

Vol 11 (1) ◽

pp. 110

Author(s):

Giulia Ricci ◽

Alessandra Cucina ◽

Sara Proietti ◽

Simona Dinicola ◽

Francesca Ferranti ◽

...

Keyword(s):

Cell Adhesion ◽

Human Keratinocytes ◽

Short Exposure ◽

Migration And Invasion ◽

Hacat Cells ◽

Invasive Phenotype ◽

Adhesion Properties ◽

Mesenchymal Transition ◽

Cell Matrix ◽

E Cadherin

Changes in cell–matrix and cell-to-cell adhesion patterns are dramatically fostered by the microgravity exposure of living cells. The modification of adhesion properties could promote the emergence of a migrating and invasive phenotype. We previously demonstrated that short exposure to the simulated microgravity of human keratinocytes (HaCaT) promotes an early epithelial–mesenchymal transition (EMT). Herein, we developed this investigation to verify if the cells maintain the acquired invasive phenotype after an extended period of weightlessness exposure. We also evaluated cells’ capability in recovering epithelial characteristics when seeded again into a normal gravitational field after short microgravity exposure. We evaluated the ultra-structural junctional features of HaCaT cells by Transmission Electron Microscopy and the distribution pattern of vinculin and E-cadherin by confocal microscopy, observing a rearrangement in cell–cell and cell–matrix interactions. These results are mirrored by data provided by migration and invasion biological assay. Overall, our studies demonstrate that after extended periods of microgravity, HaCaT cells recover an epithelial phenotype by re-establishing E-cadherin-based junctions and cytoskeleton remodeling, both being instrumental in promoting a mesenchymal–epithelial transition (MET). Those findings suggest that cytoskeletal changes noticed during the first weightlessness period have a transitory character, given that they are later reversed and followed by adaptive modifications through which cells miss the acquired mesenchymal phenotype.

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Conditioned medium from the human umbilical cord-mesenchymal stem cells stimulate the proliferation of human keratinocytes

Journal of Basic and Clinical Physiology and Pharmacology ◽

10.1515/jbcpp-2019-0283 ◽

2020 ◽

Vol 0 (0) ◽

Author(s):

Sushmitha Sriramulu ◽

Antara Banerjee ◽

Ganesan Jothimani ◽

Surajit Pathak

Keyword(s):

Stem Cells ◽

Cell Proliferation ◽

Wound Healing ◽

Mesenchymal Stem Cells ◽

Conditioned Medium ◽

Umbilical Cord ◽

Human Keratinocytes ◽

Human Umbilical Cord ◽

Hacat Cells ◽

And Migration

AbstractObjectivesWound healing is a complex process with a sequence of restoring and inhibition events such as cell proliferation, differentiation, migration as well as adhesion. Mesenchymal stem cells (MSC) derived conditioned medium (CM) has potent therapeutic functions and promotes cell proliferation, anti-oxidant, immunosuppressive, and anti-apoptotic effects. The main aim of this research is to study the role of human umbilical cord-mesenchymal stem cells (UC-MSCs) derived CM in stimulating the proliferation of human keratinocytes (HaCaT).MethodsFirstly, MSC were isolated from human umbilical cords (UC) and the cells were then cultured in proliferative medium. We prepared and collected the CM after 72 h. Morphological changes were observed after the treatment of HaCaT cells with CM. To validate the findings, proliferation rate, clonal efficiency and also gene expression studies were performed.ResultsIncreased proliferation rate was observed and confirmed with the expression of Proliferating Cell Nuclear Antigen (PCNA) after treatment with HaCaT cells. Cell-cell strap formation was also observed when HaCaT cells were treated with CM for a period of 5–6 days which was confirmed by the increased expression of Collagen Type 1 Alpha 1 chain (Col1A1).ConclusionsOur results from present study depicts that the secretory components in the CM might play a significant role by interacting with keratinocytes to promote proliferation and migration. Thus, the CM stimulates cellular proliferation, epithelialization and migration of skin cells which might be the future promising application in wound healing.

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Glycolipid-dependent and lectin-driven transcytosis in mouse enterocytes

Communications Biology ◽

10.1038/s42003-021-01693-2 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Alena Ivashenka ◽

Christian Wunder ◽

Valerie Chambon ◽

Roger Sandhoff ◽

Richard Jennemann ◽

...

Keyword(s):

Basolateral Membrane ◽

Intestinal Barrier ◽

Growth Factor Signaling ◽

Dependent Manner ◽

Galectin 3 ◽

Cell Adhesion And Migration ◽

Range Of Functions ◽

Mouse Intestine ◽

And Migration

AbstractGlycoproteins and glycolipids at the plasma membrane contribute to a range of functions from growth factor signaling to cell adhesion and migration. Glycoconjugates undergo endocytic trafficking. According to the glycolipid-lectin (GL-Lect) hypothesis, the construction of tubular endocytic pits is driven in a glycosphingolipid-dependent manner by sugar-binding proteins of the galectin family. Here, we provide evidence for a function of the GL-Lect mechanism in transcytosis across enterocytes in the mouse intestine. We show that galectin-3 (Gal3) and its newly identified binding partner lactotransferrin are transported in a glycosphingolipid-dependent manner from the apical to the basolateral membrane. Transcytosis of lactotransferrin is perturbed in Gal3 knockout mice and can be rescued by exogenous Gal3. Inside enterocytes, Gal3 is localized to hallmark structures of the GL-Lect mechanism, termed clathrin-independent carriers. These data pioneer the existence of GL-Lect endocytosis in vivo and strongly suggest that polarized trafficking across the intestinal barrier relies on this mechanism.

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